Clamping plate connection method, SpLig-HEMT biosensor, miRNA detection method and detection system

Abstract

The invention discloses a splint connection method, which accurately guides the butt joint of two nucleic acid chains through complementary splint DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) molecules and recognizes the high specificity of mononucleotide mismatch, and comprises the following steps: modifying a thiolated fixed probe on a sensing surface; the method comprises the following steps: adding a quantitative reporter probe and a fixed probe into a connection system containing SplintR ligase, a SplintR ligase reaction buffer solution and DNase / RNase-Free deionized water, and then adding a gradient diluted miRNA solution to make the total system constant in volume; incubating the SplintR ligase for a certain time at room temperature to react with alpha-phosphoric acid of ATP (adenosine triphosphate), so as to promote the release of pyrophosphoric acid and generate a covalent intermediate; the invention further discloses a corresponding SpLig-HEMT biosensor, a preparation method of the SpLig-HEMT biosensor, a rapid amplification-free miRNA detection method and a biomolecule detection system. The covalent intermediate and the 5'phosphorylation site of the reporter probe form DNA-adenylic acid, and the 3 'hydroxyl of the fixed probe attacks the DNA-adenylic acid and connects the two nucleotides.

Description

Technical Field

[0001] The present invention relates to the fields of bioinformatics, biosensors, and biomolecule detection technology, and in particular to a splint connection method, a SpLig-HEMT biosensor based on the splint connection method and a preparation method thereof, a rapid, amplification-free miRNA detection method based on the SpLig-HEMT biosensor, and a biomolecule detection system. Background Art

[0002] DNA ligases are widely used in gene cloning, modification, and ligation reactions in molecular biology. T4 DNA ligase is one of the most commonly used enzymes. It catalyzes the formation of a phosphodiester bond between a 3' hydroxyl group and a 5' phosphate group to connect nucleic acid sequences using single-stranded DNA as a template. However, when the splint molecule is RNA, the ligation efficiency of T4 DNA ligase decreases significantly, making it difficult to meet the needs of further applications. Furthermore, T4 DNA ligase typically requires a 2-4 hour reaction at 37°C, during which excess enzyme must be added to achieve good ligation efficiency. However, point-of-care (POC) assays often require room temperature, which places high demands on both time and cost. Consequently, ligation reactions represented by T4 DNA ligase have not been widely used in POC assays.

[0003] Chlorella virus DNA ligase (PBCV-1) is a small DNA ligase with only 298 amino acids, making it the smallest ligase discovered. It consists of only one nucleotide transferase and one oligonucleotide binding domain, whereas other ligases often contain N- or C-terminal DNA binding domains or other sequences of unknown function. T4 DNA ligase's ability to ligate RNA splints is primarily limited by the intermediates (adenylated phospho-ssDNA sequences). This is because ATP in the solution adenylates the ligase, which is unable to bind to the adenylated phospho-ssDNA sequences in the same active site. Consequently, the reaction requires excess enzyme and minimal ATP to proceed, but these conditions further reduce the reaction rate (Kershaw et al., 2012). However, Chlorella virus DNA ligase, with its smaller size, is more adaptable to the reaction products of different intermediates, allowing it to produce ligation products at a faster rate at high ATP concentrations. The reaction rate of PBCV-1 DNA ligase is more than a hundred times higher than that of T4 DNA ligase.

[0004] In addition to its application in the field of molecular cloning, ligase-mediated ligation reactions have also shown broad application prospects in molecular detection. By coupling the ligation reaction with the polymerase chain reaction, it can be used to detect specific nucleic acid sequences.

[0005] Ligase-based nucleic acid detection technology was first applied to the detection of DNA targets. Using specially designed probes, ligation reactions can be used to quantify gene expression levels and identify DNA copy number abnormalities, DNA methylation, and point mutation sequences. However, with the development of molecular biology, various forms of RNA have been discovered, such as small interfering RNA (miRNA), circular RNA, and modified RNA. Although RNA can currently be detected and quantified through reverse transcription, this method relies on expensive reagents and instruments and requires a long detection time, limiting its application in bedside testing. Furthermore, due to the short length of miRNA, the detection process often requires the addition of a poly(A) sequence for subsequent amplification. However, this additional step not only makes the operation more cumbersome but also increases the risk of false negatives.

[0006] A field-effect transistor (FET) is an electronic device that controls current through the electric field effect. High electron mobility field-effect transistors (HEMTs) have been widely used in biosensing. HEMTs are typically made of semiconductor materials, such as gallium nitride and gallium arsenide.

[0007] In 1969, scientists first synthesized gallium nitride (GaN) using hydride vapor phase epitaxy (HVPE). GaN can be grown on substrates such as silicon (Si), silicon carbide (SiC), and sapphire to form zincite and wurtzite crystals. The crystal structure of wurtzite GaN is composed of hexagonal unit cells, each of which contains two adjacent hexagonal close-packed sublattices. In GaN, nitrogen has a stronger electronegativity than gallium. Nitrogen atoms attract electrons from gallium atoms, resulting in an asymmetric charge distribution, making nitrogen partially negative and gallium partially positive. The wurtzite structure of GaN is non-centrosymmetric along the c-axis, with the centers of positive and negative charge not coinciding. This results in polarization along the c-axis, known as spontaneous polarization (Psp). The lattice constant of aluminum gallium nitride (AlGaN) is smaller than that of gallium nitride. When an AlGaN layer is grown on a GaN buffer layer, AlGaN will produce some tensile strain to adjust the lattice mismatch. This tensile strain then leads to the piezoelectric polarization (Ppz) of the AlGaN layer. In contrast to spontaneous polarization, piezoelectric polarization is caused by external pressure. This strain causes the crystal to deform, thereby generating a high strain-induced piezoelectric field. A heterostructure is formed between the two semiconductors AlGaN / GaN. Due to the difference in band gap energy, the conduction band (E C ) and valence band (E V) cannot be continuous at the interface. When semiconductors are brought together, the Fermi level (E f ) align, causing band bending. This band bending creates a two-dimensional potential well at the heterojunction surface, where a large number of electrons gather. These electrons can only move in two dimensions, along a plane parallel to the heterojunction, hence the name two-dimensional electron gas (2DEG). 2DEG has extremely high electron mobility.

[0008] High-electron-mobility field-effect transistor (HEMT) sensors show great potential in applications such as UV detection, gas sensing, and biosensing. UV detectors have a wide range of applications in both military and civilian fields, such as missile warning, fire warning, and environmental UV detection. Gallium nitride's wide bandgap and robustness are particularly well-suited for UV detection. In recent years, new UV detectors based on GaN have been developed, including GaN-based PIN diodes, Schottky diodes, and semiconductor-metal-based photodetectors. Gas sensors are primarily used in the automotive, aerospace, healthcare, and environmental protection sectors. In GaN-based gas sensors, gases can catalyze the dissociation of the metal gate, thereby altering the charge distribution in the channel. Oxidizing gases such as NO2 and CO2 readily acquire electrons, while reducing gases such as SO2, CO, NH3, and H2S readily lose electrons. This electron gain and loss alters the channel carrier concentration and the output drain current, which is then output as a sensing signal. High-electron-mobility transistors are also widely used in biosensing. In order to detect the analyte, the gate (sensitive area) is modified with the corresponding biosensor. When the analyte binds to the biosensor fixed on the sensing interface, the analyte will form a positive or negative charge accumulation on the sensing interface, resulting in a change in the surface potential. At the same time, the accumulated charge will cause the current in the channel to change, thereby causing the sensor drain current and threshold voltage to change. This change is output as a sensing signal.

[0009] In summary, the existing technology has the following technical defects:

[0010] 1. In the prior art method of using hybridization reaction to detect miRNA using graphene field effect transistor, the nanomaterial graphene is used as the conductive channel of the field effect transistor to prepare the graphene field effect transistor. The single-stranded DNA probe targeting miR-21 is fixed on the surface of the sensor interface through the gold-sulfur bond as a connector. Then, the target miRNA is hybridized with the DNA probe on the sensor interface at a uniform concentration, and the detection limit can be as low as 10 -20 M, while in 10 -20 M to 10 -12The sensor showed a good linear relationship at different concentrations of M and could accurately distinguish bladder cancer patients from healthy people. However, when detecting single-base mutations, the sensor showed a large number of non-specific signals, which may lead to false positive diagnoses.

[0011] 2. In addition to DNA probe molecules, Argonaute (Ago) protein, as a DNA-guided nuclease, can also recognize specific RNA sequences when guiding single-stranded DNA (gDNA). By modifying Argonaute protein on the surface of the sensing area of ​​the graphene field-effect transistor and pre-organizing it with gDNA, the binding of target miRNA to gDNA can be accelerated. The detection limit of this sensor can be as low as 10 -19 M, and can respond within 3-5 minutes. However, the manufacture of the device requires the modification of Argonaute protein and the pre-organization of gDNA in advance, which increases the complexity of the operation and limits its feasibility in clinical applications.

[0012] Therefore, it is necessary to design new biosensors and extend them to the entire biomolecule detection system to solve the problems existing in the existing technology. Summary of the Invention

[0013] To address the problems existing in the prior art, the present invention provides the following technical solutions: a splint-ligation method, a (SpLig-HEMT) biosensor based on the splint-ligation method and a preparation method thereof, a rapid, amplification-free miRNA detection method based on the SpLig-HEMT biosensor, and a biomolecule detection system. Taking advantage of the excellent properties of the AlGaN / GaN heterostructure, the SpLig-HEMT biosensor based on the splint connection method exhibits excellent transconductance performance on the order of millisiemens (mS); and in the Splint-ligation reaction step, with the help of complementary "splint" DNA or RNA molecules, the two nucleic acid chains are precisely docked. This process strictly relies on the precise matching of the target nucleotide and the splint molecule, thereby ensuring highly specific recognition of single nucleotide mismatches. This method only requires the modification of single-stranded fixed probes on the sensing surface, which simplifies the difficulty of biological fixation and lays the foundation for later clinical applications. The biomolecule detection system based on the SpLig-HEMT biosensor constructs a high-performance sensing system to form a biomolecule detection system through the ultra-sensitive response of the SpLig-HEMT sensor to the surface charge.

[0014] The first aspect of the present invention is to provide a splint ligation method that precisely guides the docking of two nucleic acid chains through complementary "splint" DNA or RNA molecules. Based on the strict reliance on the precise matching of the target nucleotide and the splint molecule, highly specific recognition of single nucleotide mismatches is achieved, comprising:

[0015] S1, thiol-modified immobilized probe on the sensing surface;

[0016] S2, adding a quantitative reporter probe and the immobilized probe to a ligation system comprising a quantitative SplintR ligase, a SplintR ligase reaction buffer solution, and DNase / RNase-free deionized water, and then adding a quantitative gradient diluted miRNA solution to adjust the total volume of the system;

[0017] S3, the SplintR ligase reacts with the α-phosphate of ATP to release pyrophosphate and generate a covalent intermediate;

[0018] S4, the intermediate forms DNA-adenylation with the 5' phosphorylation site of the reporter probe;

[0019] S5, the 3' hydroxyl group of the immobilized probe attacks the DNA-adenylate and connects two nucleotides.

[0020] Preferably, the method further comprises incubating the total system at room temperature for a first period of time after the total system is constant to volume, wherein the first period of time is 30 minutes.

[0021] Preferably, the identification is performed by gel electrophoresis analysis of the ligation product obtained by the splint ligation reaction corresponding to the splint ligation method on an agarose gel, comprising:

[0022] First, weigh a certain amount of agarose powder, add TAE solution, and heat in a microwave oven until boiling and completely dissolved;

[0023] Then, wait for it to cool to about 60°C and add nucleic acid dye and mix well;

[0024] Afterwards, the dye-added agarose solution is poured into a gelatin plate with a sample well inserted and cooled until solidified;

[0025] Finally, the prepared agarose gel was added to the electrophoresis lane, TAE buffer solution was added, 50 bp DNA marker was added as a reference, and electrophoresis was performed at 2-4 V / cm, and then analyzed using an Amersham Imager 600 gel imager.

[0026] A second aspect of the present invention is to provide a SpLig-HEMT biosensor based on the splint connection method of the first aspect, wherein the SpLig-HEMT sensor comprises:

[0027] Field effect transistor body and gate sensitive area;

[0028] Wherein, the field effect transistor body includes a source and a drain disposed on both sides;

[0029] The gate sensitive region is placed between the source and the drain, and includes a semiconductor material for forming a heterojunction structure and a bio-sensitive element probe.

[0030] Preferably, the SpLig-HEMT biosensor based on the splint connection method uses gallium nitride / aluminum gallium nitride or gallium arsenide / aluminum gallium arsenide as a heterojunction. When the analyte binds to the biosensitive element on the surface of the SpLig-HEMT biosensor, it will cause a change in surface potential. The change in surface potential is output as a signal to identify the analyte.

[0031] The third aspect of the present invention is to provide a method for preparing the SpLig-HEMT biosensor based on the splint connection method of the second aspect, comprising:

[0032] a. A silicon and gallium nitride cap layer is provided, and a buffer layer is provided between the silicon and the gallium nitride cap layer; a 1 nm thick AlN intermediate layer is provided between the gallium nitride cap layer and the AlGaN layer;

[0033] b. performing step etching to define the active area;

[0034] c. depositing a first Ti metal contact layer, an Al metal contact layer, a second Ti metal contact layer, and an Au metal contact layer by evaporation and performing rapid thermal annealing under nitrogen protection; wherein the thicknesses of the first Ti metal contact layer, the Al metal contact layer, the second Ti metal contact layer, and the Au metal contact layer are 20 nm, 110 nm, 40 nm, and 50 nm, respectively;

[0035] d. A 200nm thick SiO2 layer was deposited using plasma-enhanced chemical vapor deposition technology to isolate the interconnect layer; at the same time, a Ti metal layer, an Au metal layer, and a Ti metal layer were evaporated as the source and drain electrodes, and the source and drain electrodes were used to establish metal interconnections, wherein the thicknesses of the Ti metal layer, the Au metal layer, and the Ti metal layer were 10nm, 300nm, and 10nm, respectively;

[0036] e. Passivating the source and drain electrode surfaces using plasma-enhanced chemical vapor deposition technology, depositing 100 nm and 200 nm thick SiO2 and Si3N4 layers on the source and drain electrode surfaces, respectively; etching the SiO2 and Si3N4 layers using inductively coupled plasma and treating with a buffered fluoride oxide etchant to expose the contact pads and gate window;

[0037] f. A Ti metal layer and an Au metal layer are patterned in a 600 μm × 600 μm gate region as a sensing region. The thicknesses of the Ti metal layer and the Au metal layer are 2 nm and 10 nm, respectively. A 6 μm thick bisbenzocyclobutene material is then coated and patterned on the wafer as an insulating layer through a photolithography process, thereby forming a structure in which the GaN cap layer serves as a dielectric layer and the Au metal layer serves as a biofunctionalization layer.

[0038] Preferably, the temperature of the rapid thermal annealing is 870° C., and the annealing time of the rapid thermal annealing is 45 seconds.

[0039] The fourth aspect of the present invention is a rapid, amplification-free miRNA detection method based on the SpLig-HEMT biosensor of the second aspect, comprising:

[0040] Construction of SpLig-HEMT biosensor system;

[0041] Preparing a splint reaction system corresponding to the splint connection method; wherein the immobilized probe of the splint reaction system is pre-modified on the surface of the chip sensing area of ​​the SpLig-HEMT biosensor system, the reaction system does not contain the immobilized probe, and is filled with DNase / RNase-free deionized water;

[0042] receiving a sample to be detected, and placing the sample to be detected on the surface of the SpLig-HEMT biosensor;

[0043] It is determined that only when the reporter probe of the SpLig-HEMT biosensor and the target miRNA are present at the same time, the ligation activity of the SplintR ligase will be activated. The activated SplintR ligase connects the miRNA and the reporter probe to the fixed probe on the surface of the SpLig-HEMT biosensor, and a Splint-ligation reaction corresponding to the splint connection method occurs on the gate surface, thereby causing a change in the surface charge distribution and ultimately causing a change in the gate voltage. The current between the drain Drain and the source Source in the field effect transistor is expressed as follows:

[0044]

[0045] Where μ is the carrier mobility of SpLig-HEMT, W and L are the gate width and length respectively, C OX is the gate-to-channel capacitance, V TH 、V g and V DS are the threshold voltage, gate voltage and source-drain voltage respectively; I DSRepresents the current between the drain Drain and source Source in a field effect transistor;

[0046] miRNA-21 is detected based on the current between the drain and the source in the field effect transistor.

[0047] Preferably, the reaction system does not contain an immobilized probe, and the DNase / RNase-Free deionized water is used to fill the gap, including:

[0048] The SpLig-HEMT biosensor and the splint reaction system were incubated at room temperature for 30 minutes;

[0049] Rinse with DNase / RNase-free deionized water to remove unattached reporter probes from the chip surface.

[0050] Preferably, the construction of the SpLig-HEMT biosensor system comprises:

[0051] a. The SpLig-HEMT biosensor was treated with piranha solution and the surface of the SpLig-HEMT biosensor was cleaned with nitrogen after rinsing with deionized water;

[0052] b. To protect the thiol-modified immobilized probe from spontaneous oxidation during storage, the thiol groups are usually protected by disulfide bonds. Therefore, before modification, the immobilized probe needs to be reduced to a single chain with exposed thiol groups. Treat the immobilized probe with 1 μL of 1 mM tris(2-carboxyethyl)phosphine hydrochloride (TECP) and reduce at room temperature for 30 minutes.

[0053] c. Incubate 10 μL of the thiol-immobilized probe after reduction of TECP with the chip sensing area of ​​the SpLig-HEMT biosensor system at room temperature for 3 hours so that the fixed probe of the splint reaction system is pre-modified on the surface of the chip sensing area of ​​the SpLig-HEMT biosensor system;

[0054] d. The chip was treated with 1 M ethanolamine solution for 0.5 hours to eliminate unreacted carboxyl groups; 0.1 mg / mL bovine serum albumin solution was then added and further treated for 0.5 hours to block the remaining nonspecific binding sites on the sensing area;

[0055] e. Rinse the sensing area surface with deionized water and blow dry with nitrogen.

[0056] A fifth aspect of the present invention is to provide a biomolecule detection system based on the SpLig-HEMT biosensor of the third aspect, comprising:

[0057] The SpLig-HEMT biosensor of the third aspect of the present invention is used to output an output representing the presence and concentration of the biomolecules in the sample to be detected;

[0058] Electrical detection system, including digital source meter and integrated automation control device, used for long-term real-time monitoring of potential signals and recording of dynamic changes;

[0059] The signal processing and analysis system is used to receive the electrical signal output by the digital source meter and output characterization data of the presence state and concentration of the biological molecules of the analyte based on the processing and analysis of the electrical signal.

[0060] Preferably, the digital source meter is used for:

[0061] A. applying a high-precision bias voltage or current to the SpLig-HEMT biosensor to ensure that the field-effect transistor corresponding to the SpLig-HEMT biosensor operates stably in a linear or saturation region;

[0062] B. Measure the change in surface potential caused by the binding of biomolecules to the biosensitive elements on the surface of the SpLig-HEMT biosensor, and simultaneously measure the change in source-drain current and / or voltage, thereby converting the biological reaction of the biomolecules in the analyte to the biosensitive elements on the surface of the SpLig-HEMT biosensor into a quantifiable electrical signal;

[0063] C. Scanning the gate-source voltage and generating an output characteristic curve or a transfer characteristic curve based on the gate-source voltage; wherein the output characteristic curve or the transfer characteristic curve can be used to analyze and determine the effect of the binding of biomolecules in the analyte to the biosensitive element on the surface of the SpLig-HEMT biosensor on the threshold voltage; and the effect of the binding of biomolecules in the analyte on the threshold voltage can be determined by the offset of the output characteristic curve or the transfer characteristic curve.

[0064] Preferably, the change in surface potential is characterized by a change in at least one of source-drain current, source-drain voltage and gate-source voltage.

[0065] Preferably, the field effect transistor is a junction field effect transistor.

[0066] Preferably, the processing and analysis include:

[0067] Analyzing and determining the effect of the binding of the biomolecules in the analyte to the biosensitive element on the surface of the SpLig-HEMT biosensor on the threshold voltage through the output characteristic curve or the transfer characteristic curve; and

[0068] The presence and concentration of the biomolecules of the analyte are determined by the deviation of the output characteristic curve or the transfer characteristic curve.

[0069] The beneficial effects of the present invention include at least:

[0070] 1. The charge change after target recognition is improved. The traditional method of capturing the target through hybridization reaction using ssDNA probes only changes the charge carried by the miRNA itself. Due to its short length, the charge it carries is small. This method precisely introduces a reporter probe carrying a longer Poly (A) sequence onto the sensor surface, significantly increasing charge accumulation, thereby amplifying the response signal and improving sensitivity.

[0071] 2. Compared to hybridization methods, the Splint-ligation reaction only occurs when the miRNA sequence is fully complementary to the anchor probe and reporter probe, significantly improving specificity. In addition, the high fidelity of the SplintR enzyme enables miRNA detection at single-nucleotide resolution.

[0072] 3. The sensor can effectively distinguish between cancer patients and healthy people in the detection of clinical samples. It has excellent sensitivity and specificity in the detection of clinical samples and has broad clinical application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 This is a schematic diagram of the principle architecture of the SpLig-HEMT biosensor based on the splint connection method described in the present invention.

[0074] Figure 2 (a)- Figure 2 (f) is a flow chart of the preparation and construction method of the SpLig-HEMT biosensor provided by the present invention.

[0075] Figure 3 This is a schematic diagram of the principle architecture of the biomolecule detection system based on the SpLig-HEMT biosensor provided by the present invention.

[0076] Figure 4 (a) Figure 4 (b) and Figure 4 (c) Schematic diagram showing the sensitivity comparison between the SpLig-HEMT biosensor provided by the present invention and the hybridization method.

[0077] Figure 5 (a)- Figure 5 (d) Schematic diagrams comparing the specificity of the SpLig-HEMT biosensor and the hybridization method provided by the present invention.

[0078] Figure 6 (a)- Figure 6(e) Schematic diagrams of multiple detection results of clinical samples using the SpLig-HEMT biosensor provided by the present invention. DETAILED DESCRIPTION

[0079] In order to better understand the above technical solution, the above technical solution will be described in detail below with reference to the accompanying drawings and specific implementation methods.

[0080] The method provided by the present invention can be implemented in the following terminal environment, which may include one or more of the following components: a processor, a memory, and a display screen. The memory stores at least one instruction, which is loaded and executed by the processor to implement the method described in the following embodiments.

[0081] A processor can include one or more processing cores. It connects various components within the terminal using various interfaces and circuits. It executes instructions, programs, code sets, or instruction sets stored in memory, and accesses data stored in memory to perform various terminal functions and process data.

[0082] The memory may include random access memory (RAM) or read-only memory (ROM). The memory may be used to store instructions, programs, codes, code sets, or instructions.

[0083] The display is used to show the user interface of each application.

[0084] In addition, those skilled in the art will appreciate that the structure of the terminal described above does not limit the terminal. The terminal may include more or fewer components, or a combination of certain components, or a different arrangement of components. For example, the terminal may also include a radio frequency circuit, an input unit, a sensor, an audio circuit, a power supply, and other components, which will not be described in detail here.

[0085] Theoretical basis:

[0086] First, HEMT-based biosensors have high sensitivity and high stability and have been applied to the detection of various small molecules, nucleic acids, pathogenic microorganisms, antibodies, and tumor markers. Currently, studies have achieved glucose detection as low as 0.5nM by immobilizing glucose oxidase on HEMT sensors; and as low as 10 -15 M TNF-α detection; through the fixation of the new coronavirus spike protein antibody, 10 -22 Detection of the spike protein of M.

[0087] Second, researchers have recently proposed replacing reverse transcription with ligation. Ligation can convert challenging miRNAs into DNA sequences, enabling efficient detection through subsequent signal amplification. This approach not only simplifies the workflow but also avoids the limitations of complex equipment and reagents, enabling wider application of miRNA detection in routine laboratories and at the point-of-care.

[0088] Example 1

[0089] This embodiment provides a splint-ligation method that precisely guides the docking of two nucleic acid chains through complementary "splint" DNA or RNA molecules. Based on the strict reliance on the precise matching of the target nucleotide and the splint molecule, highly specific recognition of single nucleotide mismatches is achieved, including: S1, modifying a thiol-anchored anchor probe on the sensing surface; S2, adding a quantitative reporter probe and the anchor probe to a ligation system containing a quantitative SplintR ligase, a SplintR ligase reaction buffer solution, and DNase / RNase-free deionized water, followed by adding a quantitative gradient-diluted miRNA solution to constant the total volume; S3, the SplintR ligase reacts with the α-phosphate of ATP to promote the release of pyrophosphate and generate a covalent intermediate; S4, the intermediate forms a DNA-adenylate with the 5' phosphorylation site of the reporter probe; S5, the 3' hydroxyl group of the anchor probe attacks the DNA-adenylate and ligates the two nucleotides.

[0090] (1) In this embodiment, the specific experimental steps of the Splint-ligation reaction are as follows:

[0091] (1) Modifying the sensor surface with a thiol-anchored probe;

[0092] (2) 100 pM, 0.4 μL of the reporter probe and 100 pM, 0.4 μL of the immobilized probe were added to a ligation system containing 0.45 μL of SplintR ligase, 1 μL of SplintR ligase reaction buffer, and 5.75 μL of DNase / RNase-free deionized water. 1 μL of the serially diluted miRNA solution was then added to make a total system of 10 μL. The mixture was then incubated at room temperature for 30 minutes.

[0093] (3) The SplintR ligase reacts with the α-phosphate of ATP to release pyrophosphate and generate a covalent intermediate;

[0094] (4) the intermediate forms DNA-adenylation with the 5' phosphorylation site of the reporter probe;

[0095] (5) The 3' hydroxyl group of the immobilized probe attacks the DNA-adenylate and connects the two nucleotides.

[0096] (2) In this embodiment, the ligation product obtained by the Splint-ligation reaction needs to be identified by agarose gel.

[0097] To test the efficiency and specificity of ligation, agarose gel electrophoresis analysis was performed. Agarose gel, with its dense grid structure, can hinder the movement of nucleic acid molecules to a certain extent. In the electrophoresis solution, nucleic acid molecules are negatively charged and thus migrate toward the positive electrode in the electric field. The migration rate of nucleic acids depends on their relative molecular mass. Furthermore, gel concentration also affects the separation of nucleic acid molecules. Since this example requires the separation of 55bp and 110bp molecules, a 2% gel concentration was used:

[0098] (1) First, the reporter probe and the fixed probe quantified in step S2 are added to a ligation system comprising a quantitative SplintR ligase, a SplintR ligase reaction buffer solution, and DNase / RNase-free deionized water, and then a quantitative gradient-diluted miRNA solution is added to synchronize the total volume of the system. First, a mixed system is configured in which the specific miRNA or reporter probe is replaced with DNase / RNase-free deionized water, and gel electrophoresis analysis is performed together;

[0099] (2) In the step of incubating at room temperature for 30 minutes, the reaction time is also one of the key factors affecting the efficiency of timely detection. Therefore, in order to optimize the efficiency of the ligation reaction, the total system was incubated at room temperature for 30 minutes after the volume was fixed, and then the ligation efficiency was analyzed by gel electrophoresis.

[0100] (3) The specific operation steps of agarose gel electrophoresis analysis are as follows:

[0101] First, weigh a certain amount of agarose powder and add TAE solution (Tris-Acetate-EDTA Buffer), a buffer commonly used in molecular biology experiments. Its name comes from its main components: tris (hydroxymethylaminomethane), acetic acid, and ethylenediaminetetraacetic acid (EDTA). Heat in a microwave oven until boiling and completely dissolved.

[0102] Then, wait for it to cool to about 60°C and add nucleic acid dye and mix well;

[0103] Afterwards, the dye-added agarose solution is poured into a gelatin plate with a sample well inserted and cooled until solidified;

[0104] Finally, the prepared agarose gel was added to the electrophoresis lane, TAE buffer solution was added, 50 bp DNA marker was added as a reference, and electrophoresis was performed at 2-4 V / cm, and then analyzed using an Amersham Imager 600 gel imager.

[0105] Example 2

[0106] like Figure 1 As shown, this embodiment provides a SpLig-HEMT biosensor based on the clamping method of embodiment 1. The SpLig-HEMT sensor includes: a field effect transistor body and a gate sensitive region; wherein the field effect transistor body includes a source and a drain disposed on both sides; the gate sensitive region is disposed between the source and the drain, and includes a semiconductor material for forming a heterojunction structure and a biosensor probe.

[0107] As a preferred embodiment, the SpLig-HEMT biosensor based on the splint connection method uses gallium nitride / aluminum gallium nitride or gallium arsenide / aluminum gallium arsenide as heterojunctions. When the analyte binds to the biosensitive element on the surface of the SpLig-HEMT biosensor, it causes a change in surface potential. The change in surface potential is output as a signal to identify the analyte.

[0108] It should be noted that, in this embodiment, the SpLig-HEMT biosensor is represented by gallium nitride / aluminum gallium nitride and gallium arsenide / aluminum gallium arsenide heterojunctions, but is not limited to this type of sensor in practical applications.

[0109] Example 3

[0110] See also Figure 2 (a)- Figure 2 (f) This embodiment provides a method for preparing the SpLig-HEMT biosensor based on the splint connection method of embodiment 2, comprising:

[0111] a. A silicon and gallium nitride (GaN) cap layer is provided, and a buffer layer is provided between the silicon and the gallium nitride (GaN) cap layer; a 1 nm thick AlN (aluminum nitride) intermediate layer is provided between the gallium nitride (GaN) cap layer and the AlGaN layer;

[0112] Due to the lattice mismatch between silicon and gallium nitride (GaN), a buffer layer is required to reduce the dislocation density. In addition, the buffer layer also acts as an isolation layer between the substrate and the epitaxial layer. A 1nm thick AlN interlayer is inserted between the GaN and AlGaN layers to increase the effective conduction band offset, thereby reducing alloy scattering and improving electron mobility.

[0113] b. Perform step etching to define the active area.

[0114] c. A first Ti metal contact layer, an Al metal contact layer, a second Ti metal contact layer, and an Au metal contact layer were deposited separately by evaporation and then subjected to rapid thermal annealing under nitrogen protection; wherein the thicknesses of the first Ti metal contact layer, the Al metal contact layer, the second Ti metal contact layer, and the Au metal contact layer were 20 nm, 110 nm, 40 nm, and 50 nm, respectively; the temperature of the rapid thermal annealing was 870°C; and the annealing time of the rapid thermal annealing was 45 seconds.

[0115] d. Plasma-enhanced chemical vapor deposition technology is used to deposit a 200nm thick SiO2 layer for isolation from the interconnect layer; at the same time, a Ti metal layer, an Au metal layer, and a Ti metal layer are evaporated as the source and drain electrodes, and the source and drain electrodes are used to establish metal interconnections, wherein the thicknesses of the Ti metal layer, the Au metal layer, and the Ti metal layer are 10nm, 300nm, and 10nm, respectively.

[0116] e. Passivate the source and drain electrode surfaces based on plasma enhanced chemical vapor deposition technology, and deposit 100nm and 200nm thick SiO2 and Si3N4 layers on the source and drain electrode surfaces, respectively; etch the SiO2 and Si3N4 layers using inductively coupled plasma, and treat them with buffered fluoride oxide etchant to expose the contact pad and gate window; buffered fluoride oxide etchant (BOE) is a chemical solution based on hydrofluoric acid (HF) and ammonium fluoride (NH4F), mainly used for selective etching of oxide layers (such as SiO2, Si3N4) in semiconductor, photovoltaic and display panel manufacturing.

[0117] f. A Ti metal layer and an Au metal layer are patterned in a 600 μm × 600 μm gate region as a sensing region, with the thicknesses of the Ti metal layer and the Au metal layer being 2 nm and 10 nm, respectively. A 6 μm thick bisbenzocyclobutene material is then coated and patterned on the wafer as an insulating layer through a photolithography process, thereby forming a structure in which the gallium nitride (GaN) cap layer serves as a dielectric layer and the Au metal layer serves as a biofunctionalization layer.

[0118] In this embodiment, thanks to advances in semiconductor technology, the HEMT sensor can be very small. However, the portability of the entire system is limited by the reference electrode. The on-chip pseudo-reference electrode directly contacts the solution being measured, making it susceptible to electrochemical reactions under the influence of the electric field, thus affecting the stability of the detection system. Because this embodiment requires integrated integration, the on-chip pseudo-reference electrode was used for testing and the stability of the detection system was verified.

[0119] In this example, the stability test of the SpLig-HEMT biosensor was performed as follows: (1) First, the chip corresponding to the SpLig-HEMT biosensor was biomodified according to the method described in the previous section; (2) 10 μL of DNase / RNase-Free deionized water was added to the sensor surface of the chip and allowed to stabilize at room temperature for 10 minutes; (3) A gate voltage was then applied using an on-chip pseudo-reference electrode, and the transfer characteristic curve after the addition of DNase / RNase-Free deionized water was measured using a source meter; (4) The surface liquid was then aspirated using a pipette, and 10 μL of DNase / RNase-Free deionized water was repeatedly added, and the transfer characteristic curve after the addition was recorded; (5) The above steps were repeated 5 times, and the potential offset value was recorded to reflect the effects of manual operation and long-term testing on the stability of the device.

[0120] Example 4

[0121] This embodiment provides a rapid, amplification-free miRNA detection method based on the SpLig-HEMT biosensor of Example 2, comprising:

[0122] Construction of SpLig-HEMT biosensor system;

[0123] Preparing a splint reaction system corresponding to the splint-ligation method; wherein the immobilized probe of the splint reaction system is pre-modified on the surface of the chip sensing area of ​​the SpLig-HEMT biosensor system, the reaction system does not contain the immobilized probe, and is filled with DNase / RNase-free deionized water;

[0124] receiving a sample to be detected, and placing the sample to be detected on the surface of the SpLig-HEMT biosensor;

[0125] It was determined that only when the reporter probe of the SpLig-HEMT biosensor and the target miRNA are present at the same time, the ligation activity of the SplintR ligase will be activated. The activated SplintR ligase connects the miRNA and the reporter probe to the fixed probe on the surface of the SpLig-HEMT biosensor. Since both the reporter probe and the miRNA molecules have a phosphate backbone, they both carry negative charges, and the amount of charge increases with the length of the molecules. Therefore, a Splint-ligation reaction corresponding to the Splint-ligation method occurs on the gate surface, resulting in a change in the surface charge distribution and ultimately a change in the gate voltage (V GS ), the current between the drain Drain and the source Source in the field effect transistor is shown in the following formula (1):

[0126]

[0127] Among them, the carrier mobility of μSpLig-HEMT, W and L are the gate width and length respectively, C OX is the gate-to-channel capacitance, V TH 、V g and V DS are the threshold voltage, gate voltage and source-drain voltage respectively; I DS Represents the current between the drain Drain and source Source in a field effect transistor;

[0128] miRNA-21 is detected based on the current between the drain and the source in the field effect transistor.

[0129] As a preferred embodiment, the reaction system does not contain fixed probes, and the filling with DNase / RNase-Free deionized water includes: incubating the SpLig-HEMT biosensor and the splint reaction system at room temperature for 30 minutes; and rinsing with DNase / RNase-Free deionized water to remove unconnected reporter probes on the chip surface.

[0130] As a preferred embodiment, the construction of the SpLig-HEMT biosensor system includes:

[0131] a. First, in order to remove organic impurities in the sensing area and increase the hydrophilicity of the interface, the SpLig-HEMT biosensor was treated with piranha solution (10 minutes), and the surface of the SpLig-HEMT biosensor was rinsed with deionized water and then cleaned with nitrogen;

[0132] b. To protect the thiol-modified immobilized probe from spontaneous oxidation during storage, the thiol groups are usually protected by disulfide bonds. Therefore, before modification, the immobilized probe needs to be reduced to a single chain with exposed thiol groups. Treat the immobilized probe with 1 μL of 1 mM tris(2-carboxyethyl)phosphine hydrochloride (TECP) and reduce at room temperature for 30 minutes.

[0133] c. Incubate 10 μL of the thiol-immobilized probe after reduction of TECP with the chip sensing area of ​​the SpLig-HEMT biosensor system at room temperature for 3 hours so that the fixed probe of the splint reaction system is pre-modified on the surface of the chip sensing area of ​​the SpLig-HEMT biosensor system;

[0134] d. The chip was treated with ethanolamine solution (1 M, ie, 1 mol / L) for 0.5 hours to eliminate unreacted carboxyl groups; then bovine serum albumin solution (0.1 mg / mL) was added and further treated for 0.5 hours to block the remaining nonspecific binding sites on the sensing area;

[0135] e. Rinse the sensing area surface with deionized water and blow dry with nitrogen.

[0136] Example 5

[0137] See also Figure 3 This embodiment provides a biomolecule detection system based on the SpLig-HEMT biosensor of Example 2, comprising: the SpLig-HEMT biosensor of Example 2, configured to output data representing the presence and concentration of biomolecules in the sample to be detected; an electrical detection system, comprising a digital source meter and an integrated automation control device, configured to monitor the potential signal in real time over a long period of time and record its dynamic changes; and a signal processing and analysis system, configured to receive the electrical signal output by the digital source meter and, based on processing and analysis of the electrical signal, output data representing the presence and concentration of the biomolecules of the analyte.

[0138] As a preferred embodiment, the digital source meter is used for:

[0139] A. applying a high-precision bias voltage or current to the SpLig-HEMT biosensor to ensure that the field-effect transistor corresponding to the SpLig-HEMT biosensor operates stably in a linear or saturation region;

[0140] In this embodiment, under a high-precision bias voltage of 0.5 V, DNase / RNase-free deionized water was used as the liquid gate, and a digital source meter was used to record the transfer characteristic curves of the SpLig-HEMT biosensor before and after the reaction.

[0141] B. Used to measure the change in surface potential caused by the binding of biomolecules to the biosensitive elements on the surface of the SpLig-HEMT biosensor, and simultaneously measure the change in source-drain current and / or voltage, thereby converting the biological reaction after the biomolecules in the analyte bind to the biosensitive elements on the surface of the SpLig-HEMT biosensor into a quantifiable electrical signal.

[0142] C. Scanning the gate-source voltage and generating an output characteristic curve or a transfer characteristic curve based on the gate-source voltage; wherein the output characteristic curve or the transfer characteristic curve can be used to analyze and determine the effect of the binding of biomolecules in the analyte to the biosensitive element on the surface of the SpLig-HEMT biosensor on the threshold voltage; and the effect of the binding of biomolecules in the analyte on the threshold voltage can be determined by the offset of the output characteristic curve or the transfer characteristic curve.

[0143] As a preferred embodiment, the change in surface potential is characterized by a change in at least one of source-drain current, source-drain voltage, and gate-source voltage.

[0144] As a preferred embodiment, the field effect transistor is a junction field effect transistor (JFET).

[0145] As a preferred embodiment, the processing and analysis includes: analyzing and determining the effect of the binding of biomolecules in the analyte to the biosensitive elements on the surface of the SpLig-HEMT biosensor on the threshold voltage through the output characteristic curve or the transfer characteristic curve; and determining the presence state and concentration of the biomolecules in the analyte through the offset of the output characteristic curve or the transfer characteristic curve.

[0146] Application and beneficial effects:

[0147] 1. The charge change after target recognition is improved. The traditional method of capturing the target through hybridization reaction using ssDNA probes only changes the charge carried by the miRNA itself. Due to its short length, the charge it carries is small. This method precisely introduces a reporter probe carrying a longer Poly (A) sequence onto the sensor surface, significantly increasing charge accumulation, thereby amplifying the response signal and improving sensitivity.

[0148] like Figure 4 As shown in (a), as the concentration of miRNA-21 gradually increased (10 -19 M-10 -13M), the transfer characteristic curve of the device gradually shifts to the left, and the response signals are 2.3mV, 29.3mV, 49.6mV, 66.7mV, 85.2mV, 115.9mV and 141.7mV respectively. Based on the cut-off value of 18mV, except for the concentration of 10 -19 Except that the device does not respond at 10 M, -18 M-10 -13 The response was good within the concentration range of M.

[0149] like Figure 4 As shown in (b), similarly, the detection range and sensitivity of the HEMT biosensor platform modified with hybrid ssDNA probes of miR-21 were investigated. As the concentration of miRNA-21 increased (10 -19 M-10 -13 M), the transfer characteristic curve of the device also gradually shifted to the left, but its dynamic range and detection sensitivity were inferior to those of the SpLig-HEMT biosensor, and its signal changes were 4.2mV, 16.2mV, 36.4mV, 48.5mV, 54.5mV and 56.0mV respectively.

[0150] like Figure 4 As shown in (c), the results of the two sensors were fitted. The SpLig-HEMT -18 M-10 -13 There is a good linear relationship in the logarithmic concentration range of M, and the linear equation is Potential shift = 22.38lgC + 428.2 (R 2 =98.72%), where C is the concentration of miRNA-21. -17 A signal exceeding the cut-off value was generated at 10 -15 These experimental results show that the SpLig-HEMT biosensor of the present invention is significantly superior to the traditional ssDNA hybridization probe method in terms of dynamic range and sensitivity, demonstrating its potential in ultrasensitive detection of miRNA.

[0151] 2. Compared to hybridization methods, the Splint-ligation reaction only occurs when the miRNA sequence is fully complementary to the anchor probe and reporter probe, significantly improving specificity. In addition, the high fidelity of the SplintR enzyme enables miRNA detection at single-nucleotide resolution.

[0152] The present invention selected four heterologous miRNAs for testing (miRNA-144, miRNA-200c, miRNA-125b), and respectively added 10 -14The Splint-ligation reaction system of miRNA, an interfering molecule of M, was added to the SpLig-HEMT biosensor and incubated. Figure 5 (a) shows that the experimental results show that compared with miR-21 molecules at 10 -14 The signal change of 72.6 mV generated by the concentration of M was less than 3 times the background signal value, indicating that the SpLig-HEMT biosensor has good specificity for heterologous miRNA.

[0153] The SpLig-HEMT biosensor was then validated for its ability to recognize single-base mutations in miRNA-21 sequences. Previous studies have shown that single-stranded DNA probes exhibit low recognition rates for single-base mutations in miRNAs and are prone to generating nonspecific signals. The second to eighth nucleotides from the 5' end of a miRNA are known as the seed region. This region is crucial for miRNA binding to the 3'UTR of its target mRNA and is therefore a key structural component of miRNA regulatory function.

[0154] Therefore, in this example, two sites located in the seed region (4C>G and 7A>U) and two sites adjacent to this region (10A>U and 12A>U) were selected to evaluate whether the SpLig-HEMT biosensor can distinguish single base mutations in miRNA. Figure 5 As shown in (b), taking miRNA-21 with a single base mutation of 10A>U as an example, as the concentration of the miRNA with a single base mismatch increases from 10 -18 M increased to 10 -14 M, SpLig-HEMT did not produce a significant response signal. Figure 5 As shown in (c), ssDNA-HEMT was used as a control group. It can be seen that ssDNA-HEMT -15 M and 10 -14 A more obvious response signal was generated at M.

[0155] Further analysis such as Figure 5 As shown in (d), at 10 -18 M-10 -14 In the concentration range of 10 M, none of the four single-base mutated miRNA-21s caused a significant response in the SpLig-HEMT biosensor. -15 M concentration produced a potential shift of 30 mV, and at 10 -14 At the M concentration, a potential shift of 34.3 mV was generated, which exceeded the preset cut-off value of 18 mV.

[0156] These results demonstrate that the single-stranded DNA probe approach has significant limitations in identifying miRNAs with single-base mismatches, particularly at higher concentrations, where nonspecific signals are easily generated. In contrast, the SpLig-HEMT biosensor of the present invention effectively distinguishes single-base mutations through the Splint-ligation reaction, demonstrating excellent detection specificity and single-base resolution, providing a more reliable solution for ultrasensitive miRNA detection.

[0157] 3. Effectively distinguish cancer patients from healthy people in the detection of clinical samples

[0158] In this example, 10 serum samples from ovarian cancer patients (samples S1-S10) and 10 serum samples from healthy controls (samples H1-H10) were tested. 4 ,10 5 ,10 6 ,10 7 and 10 8 After incubation of the diluted samples, the transfer characteristic curve of the device was tested. Figure 6 As shown in (a), the sample H5 of a healthy person sample does not produce a significant response signal, and its potential offset value is between 3.07mV and 15.33mV, which does not exceed the cut-off value of 18mV. Figure 6 As shown in (b), ovarian cancer patient serum sample (S9) was tested, even at 10 8 A significant response signal of 27.57 mV appeared at all dilutions. Figure 6 As shown in (c), the sensor of the present invention is 8 -10 4 A good linear relationship was shown at various serum dilutions. Figure 6 As shown in (d), the sensing signal of each sample is presented. The potential offset value obtained from the serum sample of each ovarian cancer patient is significantly higher than the corresponding signal value of the serum sample of a healthy person, and the difference is statistically significant (p<0.0001). In addition, in order to further evaluate the detection performance of the sensor, the receiver operating characteristic (ROC) curve is drawn, as shown in Figure 2. Figure 6 As shown in (e), the area under the curve (AUC) of the sensor reached 1.0, indicating that the sensor has excellent sensitivity and specificity in clinical sample detection and has broad clinical application potential.

[0159] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic inventive concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the invention. Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the invention. Thus, the present invention is intended to include such changes and modifications as fall within the scope of the claims and their equivalents.

Claims

1. A splint connection method, characterized in that: The system precisely guides the docking of two nucleic acid chains through complementary "splint" DNA or RNA molecules, and recognizes single nucleotide mismatches with high specificity based on the precise match between the target nucleotide and the splint molecule, including: S1, thiol-modified immobilized probe on the sensing surface; S2, adding a quantitative reporter probe and the immobilized probe to a ligation system comprising a quantitative SplintR ligase, a SplintR ligase reaction buffer solution, and DNase / RNase-free deionized water, and then adding a quantitative gradient diluted miRNA solution to adjust the total volume of the system; S3, the SplintR ligase reacts with the α-phosphate of ATP to release pyrophosphate and generate a covalent intermediate; S4, the covalent intermediate forms DNA-adenylation with the 5' phosphorylation site of the reporter probe; S5, the 3' hydroxyl group of the immobilized probe attacks the DNA-adenylate and connects two nucleotides.

2. A splint connection method according to claim 1, characterized in that: The method further includes incubating the total system at room temperature for a first period of time after the total system is adjusted to volume, wherein the first period of time is 30 minutes.

3. A splint connection method according to claim 2, characterized in that: The identification is performed by gel electrophoresis analysis of the ligation product obtained by the splint ligation reaction corresponding to the splint ligation method through agarose gel, comprising: First, weigh a certain amount of agarose powder, add TAE solution, and heat in a microwave oven until boiling and completely dissolved; Then, wait for it to cool to about 60°C and add nucleic acid dye and mix well; Afterwards, the dye-added agarose solution is poured into a gelatin plate with a sample well inserted and cooled until solidified; Finally, the prepared agarose gel was added to the electrophoresis lane, TAE buffer solution was added, 50 bp DNA marker was added as a reference, and electrophoresis was performed at 2-4 V / cm, and then analyzed using an Amersham Imager 600 gel imager.

4. A SpLig-HEMT biosensor based on the splint connection method according to any one of claims 1 to 3, characterized in that: The SpLig-HEMT biosensor comprises: Field effect transistor body and gate sensitive area; Wherein, the field effect transistor body includes a source and a drain disposed on both sides; The gate sensitive region is placed between the source and drain, and includes semiconductor materials for forming a heterojunction structure and modified with bio-sensitive element probes.

5. The SpLig-HEMT biosensor according to claim 4, characterized in that The SpLig-HEMT biosensor based on the splint connection method uses gallium nitride / aluminum gallium nitride or gallium arsenide / aluminum gallium arsenide as a heterojunction. When the analyte binds to the biosensitive element on the surface of the SpLig-HEMT biosensor, it causes a change in surface potential. This change in surface potential is output as a signal to identify the analyte.

6. The method for preparing a SpLig-HEMT biosensor based on the splint connection method according to any one of claims 4-5, characterized in that: include: a. Providing a silicon and gallium nitride cap layer, and providing a buffer layer between the silicon and the gallium nitride cap layer; Disposing a 1 nm thick AlN intermediate layer between the gallium nitride cap layer and the AlGaN layer; b. performing step etching to define the active area; c. depositing a first Ti metal contact layer, an Al metal contact layer, a second Ti metal contact layer, and an Au metal contact layer by evaporation and performing rapid thermal annealing under nitrogen protection; wherein the thicknesses of the first Ti metal contact layer, the Al metal contact layer, the second Ti metal contact layer, and the Au metal contact layer are 20 nm, 110 nm, 40 nm, and 50 nm, respectively; d. Plasma-enhanced chemical vapor deposition is used to deposit a 200nm thick SiO2 layer for isolation from the interconnect layer; simultaneously, a first Ti metal layer, a first Au metal layer, and a second Ti metal layer are evaporated as source and drain electrodes, the source and drain electrodes being used to establish metal interconnections, wherein the thicknesses of the first Ti metal layer, the first Au metal layer, and the second Ti metal layer are 10nm, 300nm, and 10nm, respectively; e. Passivating the source and drain electrode surfaces using plasma-enhanced chemical vapor deposition technology, depositing 100 nm and 200 nm thick SiO2 and Si3N4 layers on the source and drain electrode surfaces, respectively; etching the SiO2 and Si3N4 layers using inductively coupled plasma and treating with a buffered fluoride oxide etchant to expose the contact pads and gate window; f. Patterning a third Ti metal layer and a second Au metal layer in a 600 μm × 600 μm gate region as a sensing region, wherein the thicknesses of the third Ti metal layer and the second Au metal layer are 2 nm and 10 nm, respectively; and coating and patterning a 6 μm thick bisbenzocyclobutene material on the wafer as an insulating layer through a photolithography process, thereby forming a structure in which the gallium nitride cap layer serves as a dielectric layer and the Au metal layer serves as a biofunctionalization layer.

7. The preparation method according to claim 6, characterized in that The temperature of the rapid thermal annealing is 870° C., and the annealing time of the rapid thermal annealing is 45 seconds.

8. A rapid, amplification-free miRNA detection method based on the SpLig-HEMT biosensor according to any one of claims 4-5, characterized in that: include: Construction of SpLig-HEMT biosensor system; Preparing a splint reaction system corresponding to the splint connection method; wherein the immobilized probe of the splint reaction system is pre-modified on the surface of the chip sensing area of ​​the SpLig-HEMT biosensor system, the reaction system does not contain the immobilized probe, and is filled with DNase / RNase-free deionized water; receiving a sample to be detected, and placing the sample to be detected on the surface of the SpLig-HEMT biosensor; It is determined that only when the reporter probe of the SpLig-HEMT biosensor and the target miRNA are present at the same time, the ligation activity of the SplintR ligase will be activated. The activated SplintR ligase connects the miRNA and the reporter probe to the fixed probe on the surface of the SpLig-HEMT biosensor, and a splint connection reaction corresponding to the splint connection method occurs on the gate surface, thereby causing a change in the surface charge distribution and ultimately causing a change in the gate voltage. The current between the drain Drain and the source Source in the field effect transistor is expressed as follows: Where μ is the carrier mobility of SpLig-HEMT, W and L are the gate width and length respectively, C OX is the gate-to-channel capacitance, V TH 、V g and V DS are the threshold voltage, gate voltage, and source-drain voltage respectively; I DS Represents the current between the drain Drain and source Source in a field effect transistor; miRNA-21 is detected based on the current between the drain and the source in the field effect transistor.

9. The rapid, amplification-free miRNA detection method according to claim 8, characterized in that: The construction of the SpLig-HEMT biosensor system comprises: a. The SpLig-HEMT biosensor was treated with piranha solution and the surface of the SpLig-HEMT biosensor was cleaned with nitrogen after rinsing with deionized water; b. To protect the thiol-modified immobilized probe from spontaneous oxidation during storage, the thiol groups are usually protected by disulfide bonds. Therefore, before modification, the immobilized probe needs to be reduced to a single chain with exposed thiol groups. Treat the immobilized probe with 1 μL of 1 mM tris(2-carboxyethyl)phosphine hydrochloride (TECP) and reduce at room temperature for 30 minutes. c. Incubate 10 μL of the thiol-immobilized probe after reduction of TECP with the chip sensing area of ​​the SpLig-HEMT biosensor system at room temperature for 3 hours so that the fixed probe of the splint reaction system is pre-modified on the surface of the chip sensing area of ​​the SpLig-HEMT biosensor system; d. The chip was treated with 1 M ethanolamine solution for 0.5 hours to eliminate unreacted carboxyl groups; 0.1 mg / mL bovine serum albumin solution was then added and further treated for 0.5 hours to block the remaining nonspecific binding sites on the sensing area; e. Rinse the sensing area surface with deionized water and blow dry with nitrogen.

10. The rapid, amplification-free miRNA detection method according to claim 9, characterized in that: The reaction system does not contain fixed probes and is supplemented with DNase / RNase-free deionized water, including: The SpLig-HEMT biosensor and the splint reaction system were incubated at room temperature for 30 minutes; Rinse with DNase / RNase-free deionized water to remove unattached reporter probes from the chip surface.

11. A biomolecule detection system, characterized in that: include: The SpLig-HEMT biosensor according to any one of claims 4-5, which is used to output an output representing the presence and concentration of biomolecules in a sample to be detected; Electrical detection system, including digital source meter and integrated automation control device, used for long-term real-time monitoring of potential signals and recording of dynamic changes; The signal processing and analysis system is used to receive the electrical signal output by the digital source meter and output characterization data of the presence state and concentration of the biological molecules of the analyte based on the processing and analysis of the electrical signal.

12. The biomolecule detection system according to claim 11, characterized in that: The digital source meter is used to: A. applying a high-precision bias voltage or current to the SpLig-HEMT biosensor to ensure that the field-effect transistor corresponding to the SpLig-HEMT biosensor operates stably in a linear or saturation region; B. Measure the change in surface potential caused by the binding of biomolecules to the immobilized probe elements on the surface of the SpLig-HEMT biosensor, and simultaneously measure the change in source-drain current and / or voltage, thereby converting the binding of biomolecules in the analyte to the biosensitive elements on the surface of the SpLig-HEMT biosensor into a quantifiable electrical signal; C. Scanning the gate-source voltage and generating an output characteristic curve or a transfer characteristic curve based on the gate-source voltage; wherein the output characteristic curve or the transfer characteristic curve can be used to analyze and determine the effect of the binding of biomolecules in the analyte to the biosensitive element on the surface of the SpLig-HEMT biosensor on the threshold voltage; and the effect of the binding of biomolecules in the analyte on the threshold voltage can be determined by the offset of the output characteristic curve or the transfer characteristic curve.

13. A biomolecule detection system according to claim 12, characterized in that: The change in surface potential is characterized by a change in at least one of a source-drain current, a source-drain voltage, and a gate-source voltage.

14. A biomolecule detection system according to claim 13, characterized in that: The field effect transistor is a junction field effect transistor.

15. The biomolecule detection system according to claim 14, characterized in that: The processing and analysis include: Analyzing and determining the effect of the binding of the biomolecules in the analyte to the biosensitive element on the surface of the SpLig-HEMT biosensor on the threshold voltage through the output characteristic curve or the transfer characteristic curve; and The presence and concentration of the biomolecules of the analyte are determined by the deviation of the output characteristic curve or the transfer characteristic curve.

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